Nascent peptides are short amino acid sequences that emerge from the peptidyl transferase reaction center during the early stages of protein biosynthesis in the ribosome. Some nascent peptides in prokaryotes contain special sequence motifs that act in cis and regulate the expression of antibiotic resistance genes by a mechanism that involves ribosomal stalling. The proposed research will establish the structural basis of nascent peptide-induced antibiotic resistance in the exit tunnel of the ribosome. The research plan involves the use of a multidisciplinary approach that combines synthetic organic chemistry, biochemistry and structural biology. We will synthesize specific nascent peptide sequences and covalently link them to peptidyl transferase inhibitors for their delivery into the peptidyl transferase reaction center and the ribosomal exit tunnel for analysis. We will define the mechanism and mode of interactions of these sequence motifs with the exit tunnel and specific antibiotics at atomic resolution. The results will provide fundamental insights into the mechanism of antibiotic resistance and will produce a broad impact on rational drug design and development.